Abstract
The study of mitochondrial DNA (mtDNA) integrity and how replication, transcription, repair, and degradation maintain mitochondrial function has been hampered due to the inability to identify mtDNA structural forms. Here we describe the use of 2D intact mtDNA agarose gel electrophoresis, or 2D-IMAGE, to identify up to 25 major mtDNA topoisomers such as double-stranded circular mtDNA (including supercoiled molecules, nicked circles, and multiple catenated species) and various forms containing single-stranded DNA (ssDNA) structures. Using this modification of a classical 1D gel electrophoresis procedure, many of the identified mtDNA species have been associated with mitochondrial replication, damage, deletions, and possibly transcription. The increased resolution of 2D-IMAGE allows for the identification and monitoring of novel mtDNA intermediates to reveal alterations in genome replication, transcription, repair, or degradation associated with perturbations during mitochondrial stress.
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Acknowledgments
This work was support by NIH grants GM110424, MH119335 and HD099666 to B.A.K. The authors wish to recognize the efforts of Petra Langer-Cravens, Ph.D. who provided writing assistance for the manuscript.
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Kolesar, J.E., Kaufman, B.A. (2020). Using Two-Dimensional Intact Mitochondrial DNA (mtDNA) Agarose Gel Electrophoresis (2D-IMAGE) to Detect Changes in Topology Associated with Mitochondrial Replication, Transcription, and Damage. In: Hanada, K. (eds) DNA Electrophoresis. Methods in Molecular Biology, vol 2119. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0323-9_3
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DOI: https://doi.org/10.1007/978-1-0716-0323-9_3
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